Extraction of phytosterols from corn fiber using green solvents

ABSTRACT

The present invention relates to the use of “green” or relatively benign solvents such as ethanol, ethanol/water, isopropyl alcohol, isopropyl alcohol/water, ethyl lactate, acetone, butanol, isoamyl alcohol, or ethyl acetate to extract phytosterols from wet corn fiber. The resulting oil product contains free phytosterols and free fatty acids.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/365,816, filed Mar. 21, 2002, the content of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of “green” or relatively benignsolvents such as ethanol, ethanol/water, isopropyl alcohol, isopropylalcohol/water, ethyl lactate, acetone, butanol, isoamyl alcohol, orethyl acetate to extract phytosterols from wet corn fiber.

2. Background Art

Cholesterol is an important part of every human's daily diet because itis needed in the body for certain important functions, such as formingnerve cell membranes and aiding the production of hormones. Normally,cholesterol is absorbed in the small intestine and is eventually carriedby special lipid and protein complexes called Low Density Lipoproteins(“LDL”). LDL delivers cholesterol to the tissues via the blood and cellsthat need cholesterol express specific, external LDL receptors. HighDensity Lipoproteins (“HDL”) collect cholesterol from dying cells andmembranes undergoing turnover for recycling. The ratio between LDL andHDL in the bloodstream can be used to predict a patient's health status.If the ratio is above 3.5, then the patient has an elevated LDL or bloodcholesterol level. (Biochemistry, 5th Edition, Berg, J. M., et al.,eds., W. H. Freeman & Co, New York, N.Y., pp. 726-731 (2002)).

The amount of cholesterol necessary to carry out these functions is,unfortunately, only a tiny portion of the daily cholesterol intake of anaverage American. When excess cholesterol is ingested or when there is abreakdown in the processing of cholesterol from LDL, it can lead toartherosclerosis or clogged arteries. If an artery to the heart isblocked by an artherosclerotic plaque, it can cause a heart attack.Similiar blockage of arteries in the brain can lead to strokes. To lowerthe risk of a patient having a heart attack or stroke, the bloodcholesterol level must be lowered (WO 01/15552). The most direct routeto manage blood cholesterol is by lowering dietary intake. There areonly a few drugs on the market that inhibit bile recycling or de novocholesterol biosynthesis (<<http://nh1bisupport.com/chd1/meds.htm>>,National Heart, Lung, and Blood Institute, (2002)).

Phytosterols, or plant fats that include beta-sitosterol and itsglucoside beta-sitosterolin, closely resemble the molecule cholesterol(Hicks and Moreau, Food Technology 55(1):63-67 (2001)). Since themid-1960's, over 5,000 research articles have been published on thepositive benefits of phytosterols, including the fact that theyinterfere with cholesterol absorption. The lowered absorption ofcholesterol from the intestines decreases the LDL, lowering plasmacholesterol levels (Nigon, et al., Sang Thrombose Vaisseaux12(8):483-490 (2000)).

Phytosterols have been found to be present in corn fiber, which is aby-product obtained from “wet-milling” corn (Moreau, et al., BiochemicalSociety Transactions 28(6):803-806 (2000)). “Wet-milling” is a processby which corn can be separated into its basic components: starch,protein, oil, and fiber (<http://www.corn.org/web/process.htm>, CornRefiners Association, (2002)).

“Corn fiber” is defined in U.S. Pat. No. 5,843,499 as “the productobtained from the wet-milling process, which involves an initialsteeping of corn kernels in aqueous sulfur dioxide at an elevatedtemperature followed by gentle grinding and physical separation of theouter fiber layers from starch, protein and other components” (see alsoSingh, et al, Cereal Chem. 77(5):665-668 (2000)). For the purpose ofthis invention, corn fiber will have the meaning just described.

Corn fiber is produced by corn wet-milling at the rate of ˜11% perbushel of corn processed. This means that over 14,000 tons of corngluten feed and corn oil meal are produced per day, with corn oil mealbeing a very small fraction (less than 680 tons per day)(<<http://corn.org/web/shipprod. htm>>, Corn Refiners Association,(2003)). Currently, corn fiber is a low value waste stream that, withthe addition of protein, can be sold as corn gluten (animal) feed (U.S.Pat. No. 4,038,481). In an effort to upgrade this co-product and harvestphytosterols, a cost-effective strategy was developed. To this end, asolvent system compatible with current corn wet-milling operations isimportant.

According to Moreau et al. oils containing phytosterols can be extractedfrom corn fiber using hexanes with the antioxidant BHT present. TheMoreau procedures were completed on dried, ground corn fiber at roomtemperature with agitation. This extraction resulted in mixed oilscontaining triglycerides (TAG), fatty acid esters of phytosterols(St-FA), free fatty acids (FFA), tocopherols, free phytosterols (St),and ferulic acid esters of phytosterols (St-F). Moreau reportedapproximately 15% (wt/wt) sterol content with about 7% as St-FA (U.S.Pat. No. 5,843,499).

Moreau's method differs significantly from the present invention in thatthe extractions described herein may be carried out on either dry cornfiber or wet, unground corn fiber. An advantage to the present inventionis that the corn fiber need not be dried or ground. While some reductionof water may be necessary, the lack of need for completely dry cornfiber is an advantage because the energy needed to reduce the fiber from65% water (typical for wet-milled corn fiber) to 0% water is high.Additionally, grain dust explosions are a huge potential hazard forgrain storage and milling operations. This hazard is greatly reduced bynot grinding the corn fiber to the 20 mesh grind that the '499 patentsuggests. By processing wet corn fiber with larger particle sizes thechance of a grain dust explosion is minimized.

The present invention also differs from the process outlined in the '499patent because the phytosterols isolated using the method of the presentinvention are isolated as free sterols, not as a mixture of sterylferulates, steryl fatty acid esters, and free sterols as seen by Moreau.The present invention provides for both the selective and totalextraction of phytosterols based upon the extraction solvent and watercontent. The saponification conditions applied in the present inventionare effective at reducing the oil to two major components: free fattyacids and free sterols.

One goal of the present invention was to optimize a cost-effectivemethod of selectively extracting phytosterols from corn fiber using anenvironmentally friendly solvent, which is an improvement on the currenttechnology. Green solvents are less toxic than common organic solvents,like hexane (Hanmaoungjai, P., et al., J. Am. Oil Chem. Soc.78(8):817-821 (2001)). The term “green solvents” in the presentdisclosure will be considered to comprise water, ethanol, isopropylalcohol, ethyl lactate, acetone, butanol, isoamyl alcohol, or ethylacetate and a blend of one or more thereof. These solvents degrade morerapidly in the environment, are less toxic to mammals than many othersolvents, and are consistent with the twelve principles of “greenchemistry” (<<http://chemistry.org/portal/Chemistry?PID=acsdisplay.html&DOC=education\greenchem\principles.html>>, The AmericanChemical Society, (2002)).

Ethanol and ethanol/water mixtures are most compatible with current cornwet-milling plants since both ethanol and water are available for use insuch plants. The inventors of the present invention have found that pureethanol extracts have the same yield of corn fiber oil as if extractedby hexane.

Organic solvents do not selectively extract only phytosterols, which isin contrast to the solvents used in the present invention. It is acommon known practice to those skilled in the art that extraction ofphytosterols can be done by reacting dried, ground corn fiber withorganic solvents, such as hexane, chloroform, ether, and methanol.However, these organic solvents extract a mixture of components from thecorn fiber, including triglycerides, and therefore the final isolationof the phytosterols from the triglycerides and further purification bycrystallization is further complicated.

Purifying a corn lipid extract using crystallization is disclosed inU.S. Pat. No. 6,352,845. The '845 patent discloses an extraction of acorn fiber lipid fraction from wet or dry unground corn fiber using asolvent. After the solvent is removed from the corn fiber lipidfraction, phytosterols and phytosterol esters can be isolated from thecorn fiber lipid fraction in a mixture or as individual fractions bycrystallization.

WO 00/69280 teaches using ethanol (a green solvent) to extract total oiland zein (protein) from dry-milled corn residue (or corn bran). This isdifferent from the present invention because the starting material for00/69280 is corn bran, which is a byproduct of dry milling, rather thancorn fiber, which is derived from corn wet milling. Corn fiber is abetter source of phytosterols than corn bran because corn fiber containsless oil and has a higher phytosterol content. The present invention hasoptimized the method of selectively extracting the total sterols fromcorn without any triglycerides or proteins in the final oil product. Thewet corn fiber used in the present invention can be taken directly froma wet mill and reacted with a green solvent to start the presentdisclosed invention.

SUMMARY OF THE INVENTION

The present invention relates to extracting phytosterols from corn fiberusing an environmentally friendly solvent, wherein the corn fiber neednot be dried or ground. The present invention has optimized the methodof selectively extracting the total sterols from corn fiber without anytriglycerides or proteins in the final oil product. The wet corn fiberused in the present invention can be taken directly from a wet mill andreacted with a green solvent to start the present disclosed invention.

The solvents to be used in the present invention can be selected fromthe group of green solvents comprising water, ethanol, isopropylalcohol, ethyl lactate, acetone, butanol, isoamyl alcohol, ethyl acetateand a blend of one or more thereof. Most experiments disclosed hereinwere completed with a mixture of ethanol and water. Many ratios ofethanol to water were evaluated as to the effectiveness of extractingthe phytosterols. In a preferred embodiment, the ratio of ethanol towater is 80% ethanol/20% water.

In another preferred embodiment, the corn fiber and solvent mixture isagitated and heated. After agitating and heating the mixture, the solidparticles were removed by filtration.

In most cases, direct treatment with base (10% or 1% NaOH or KOH)degraded any triglycerides, steryl fatty acid esters, and/or sterylferulate esters that were extracted. Heating enhanced the reaction ratefor saponification and created free fatty acids, fatty acid esters, freesterols, free stanols, and other small molecules (like ferulic acid).

In one embodiment, the supernatant of this filtration was distilled toremove the majority of the solvent by simple, vacuum distillation. Thewater-soluble components of the resulting mixture were removed aftersaponification (base treatment) by extracting twice with hexane. Theremainder of the water was then removed by drying with MgSO₄ and thehexane was removed by simple, vacuum distillation. This resulted in afinal, phytosterol-rich oil.

One goal of the present invention was to optimize a cost-effectivemethod of selectively extracting phytosterols from corn fiber using anenvironmentally friendly solvent, which is an improvement on the currenttechnology. Another advantage to the present invention is that the cornfiber need not be dried or ground. The present invention has optimizedthe method of selectively extracting the total sterols from corn withoutany triglycerides or proteins in the final oil product.

The wet corn fiber used in the present invention can be taken directlyfrom a wet mill and reacted with a green solvent to start the presentdisclosed invention. The use of wet-milled corn fiber aids processintegration with an existing wet mill operation by reducing the numberof steps required to obtain the phytosterols and saving the energy thatis normally required to dry the corn fiber (Corn: Chemistry andTechnology, Watson, S. A. et al., eds., American Association of CerealChemicals, Inc., St. Paul, Minn., p.384 (1987)). Additional savings arealso realized because of the reduced energy required for the defattedfiber. Less drying is required for the post-solvent treatment aftertreating the corn fiber with a solvent. This is due to the fact that theextraction aids in significant water removal. In the currently existingwet mills, the corn fiber stream that results from the process accordingto the present invention can be combined with evaporated steepwater, andthen dried and sold as corn gluten feed. An advantage of the presentinvention is that whether the water originates from wet fiber or addedto dry fiber with the solvent, as in one embodiment using anethanol/water mixture, the results of the extraction are the same.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sterol compositions for Samples 111A-4195, 111C-4195,111D-4195, and 111E-4195.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, it was determined that when green solvents aremixed with corn fiber, sterols can be selectively extracted withoutextracting any triglycerides. It was determined that this could be donewith native or destarched corn fiber. Accordingly, both native anddestarched corn fiber can be used in the present invention. For thepurpose of this disclosure, “native corn fiber” will be considered ascorn fiber that was taken directly from the corn wet-milling processand, without further manipulation, was used as starting material for thepresent invention. “Destarched corn fiber” is corn fiber that has beentaken directly from the corn wet-milling process and was enzymaticallydestarched. It was found that destarching the corn fiber beforebeginning the process as described in the present invention resulted ina final oil with a higher percentage of total sterols.

For the purposes of the present invention, “phytosterols” will beassumed to include beta-sitosterol, sitostanol, campesterol,campestanol, stigmasterol, stigmastanol, brassicasterol, and othercompounds containing the sterol ring system. In this disclosure, “totalsterols” includes all of the phytosterols described above. Phytosterolswill also be assumed to include sterol glucosides, sterol fatty acidesters, and sterol ferulate esters.

As mentioned above, the green solvent to be used in the presentinvention can be, for example, water, ethanol, isopropyl alcohol, ethyllactate, acetone, butanol, isoamyl alcohol, ethyl acetate or a blend ofone or more thereof. Most experiments of the present invention were donewith a mixture of ethanol and water rather than only ethanol foreconomic reasons. Wet corn fiber directly from the wet-milling processis approximately 65% water. The method was successfully carried outusing corn fiber directly from the wet milling process; however, thedisadvantage of having corn fiber with 65% water was that to achieve an80% ethanol/20% water mixture, an extreme amount of anhydrous ethanolhad to be added. The method was optimized by first removing some of thiswater from the corn fiber by vacuum drying at 80° C. The water wasremoved until the corn fiber was ˜20% water. This made the volumesnecessary to achieve the 80/20 ratio of ethanol to water moremanageable. Many other ratios of ethanol to water were attemptedsuccessfully and are within the scope of the present invention; 80/20gave the best results in terms of selectively extracting phytosterols.The use of wet corn fiber is an important concept because it aids inimproved corn wet milling process economics by integration into existingwet mills with the minimum number of steps. The removal of completedrying results in energy cost savings, which makes the process ofphytosterol extraction more economically attractive.

In the present invention, it was determined that agitating thedestarched, wet corn fiber and ethanol mixture was preferred forisolation of the phytosterols. The method was completed withoutagitating and phytosterols were selectively extracted. However, thepercentage of total sterols in the final oil without agitation was muchlower than when agitation was utilized. Once it was determined thatagitation was beneficial, the degree of agitation was optimized. Themethod was tested with up to 1000 revolutions per minute (rpm) ofagitation. In a preferred embodiment, the mixture is agitated at 500rpm.

Heating the mixture of wet corn fiber and ethanol was also utilized tooptimize the amount of total sterols extracted. The mixture was heatedwith temperatures ranging from 25-50° C. In a preferred embodiment, themixture is heated to a temperature of 50° C.

After agitating and heating the mixture, the solid particles can beremoved. Filtration methods, which would be apparent to those skilled inthe art include, but are not limited to, centrifugation, screw press,twin roll press and vacuum filtration with a porous screen.

Base-catalyzed saponification degraded any triglycerides, steryl fattyacid esters, and/or steryl ferulate esters that were extracted. Heatingto 50° C. enhanced the reaction rate and created free fatty acids, fattyacid esters, free sterols, free stanols, and other small molecules (likeferulic acid). Sodium hydroxide and potassium hydroxide (1-10% wt/v)were explored. The results favor the use of 1% sodium hydroxide orcaustic due to the minimum formation of salt (ash). The use of 1% NaOHalso appears to aid the maximum removal of other co-extractants, such asdiferuloylputrescine and coumaroylferuloylputrescine (Moreau et al.,Lipids 36(8):839-844 (2001)).

The pH 2-4 product mixture was distilled to remove the majority of theethanol. The inventors of the present invention attempted rotaryevaporation, simple distillation and simple, vacuum distillation withthe latter of the three being the preferred method. The supernatant wasdistilled to remove the ethanol, leaving anywhere from ⅓ to ¼ of theinitial volume, with ¼ being preferred.

In one embodiment, the water-soluble components of the above resultingmixture are removed by treating the mixture with two times the aboveresulting volume of hexane. The hexane was dried with MgSO₄, thenremoved. Again, rotary evaporation, simple distillation and simple,vacuum distillation were all attempted. Simple, vacuum distillation wasthe preferred method. The final product was a phytosterol-rich oil.

EXAMPLES Example 1 Solvent Study

This study compared solvents for selective extraction of phytosterolsfrom corn fiber and was monitored by thin layer chromatography (TLC)using 95:5:1 (v/v/v) hexanes:isopropanol:acetic acid on silica gel withan acidic ferric chloride dip that, after heating, assists in visuallydetecting sterols and other lipid components. Sterols (St), sterylferulates (St-F) and sterol fatty acid esters (St-FA) have a 0.1 mgdetection limit, while the detection limit for free fatty acids (FFA) is100 mg in this methodology.

Dry, ground, destarched corn fiber (DCF; ˜4 g) was placed in six glassscrewcap tubes. The solvent (40 mL) indicated in Table 1 was placed inthe tube with the DCF. The tubes were incubated at 25° C. for 1 hour and42° C. for 1 hour with agitation (200 rpm). Extracts were removed byfiltration and the solvent was evaporated under reduced pressure. TLCwas completed on these samples with standards present on the same plate.

TABLE 1 A preliminary study of the relationship between solvent andextractable lipid components in destarched corn fiber. % TLCObservations (most Sample Solvent Water abundant to least abundantlipids) 18A-4195 Ethanol 25 FFA, St, St-F, TAG 18B-4195 Ethanol 10 FFA,St, St-F, TAG 18C-4195 Isopropanol 10 TAG, FFA, St, St-F, St-FA 18D-4195Hexanes 0 TAG, FFA, St, St-F, St-FA 18E-4195 Ethyl 0 TAG, FFA, St, St-F,St-FA Acetate 18F-4195 Ethanol 0 TAG, FFA, St, St-F, St-FA

The typical composition of corn fiber oil is approximately 80 wt %triglycerides (TAG), yet the TLC data indicate that when ethanol/waterwas used as the solvent for extraction this was not the case. This studydemonstrated that ethanol/water mixtures did not extract the same lipidcomponents in the same quantitative distribution as other solvents suchas hexanes and ethyl acetate.

Example 2 Ethanol/Water Selectivity Study

This experiment was designed to investigate phytosterol extraction usingdifferent concentrations of water in ethanol.

Dry, ground, DCF (˜8 g) was placed in Erlenmeyer flasks with 80 mL ofethanol/water. Each flask had a different ethanol/water mixture. Thesamples were immersed in the extractant for 2 hours at 25° C. and wereextracted at 42° C. for two additional hours without agitation. Thesamples were filtered, and the solvent was removed by vacuumdistillation, and desiccated. The lipid contents of these samples aredescribed in Tables 2 and 3.

TABLE 2 A preliminary study of the relationship between water contentand the ethanol extractable lipid components in DCF. % TLC Observations(most abundant Sample Solvent Water to least abundant lipids) 80A-4195Ethanol 5 TAG, FFA, St, St-F, St-FA 80B-4195 Ethanol 10 TAG, FFA, St,St-F, St-FA 80C-4195 Ethanol 15 TAG, FFA, St, St-F, St-FA 80D-4195Ethanol 20 FFA, St, St-F, TAG 80E-4195 Ethanol 25 FFA, St, St-F

These samples (80A-4195 through 80E-4195) contained significant nonlipidimpurities as evidenced by large losses (˜65%) during saponificationreactions, making complete mass balance particularly challenging. Thesefindings confirmed a recent paper by Moreau in Lipids that reportsethanol solvents will extract putrescine diesters as well as othernonlipid materials (Moreau, Robert A., et al., Lipids 36:839-844(2001)). Reported in Table 3 are the total FFA and total St for eachsaponified sample. During saponification all St-FA and St-F wereconverted into St, and TAG was converted, predominantly, into FFAs.

TABLE 3 GC results for saponified, silylated DCF extracts obtained usingdifferent ethanol/water concentrations. % Water in Total FFA, TotalSterols, Sample ethanol wt % wt % 80A-4195 5 63 8 80B-4195 10 32 880C-4195 15 39 13 80D-4195 20 24 10 80E-4195 25 4 7

Both the total sterol content and the total free fatty acid content ofthe extract were impacted by the amount of water present in the ethanol.With only 5% water in ethanol, the FFA number is high and the sterolnumber was low, whereas with 15-20% water, sterols made up a little morethan 10% of the sample. The lower the water content in ethanol, the moreit extracted the same lipid components as hexane. There also appeared tobe a solvent polarity range that is selective for sterols, since thepeak sterol concentration is near 85% ethanol and 15% water.

Wet DCF (100 g; 64% water) was extracted with ethanol/water (600 mL) at47° C. for two hours. The fiber was filtered while hot and the extractwas subjected to solvent reduction via vacuum distillation followed byhexane/salt-water extraction. The hexane layer was dried with magnesiumsulfate, filtered, and then distilled to crude lipids.

Tables 4 and 5 contain the results of both TLC and gas chromatography(GC) for Samples 111A-4195 , 111C-4195, 111D-4195, and 111E-4195. At andabove 15% water, the initial lipid extracts indicate selectivity towardFFA and away from TAG, thus enhancing the sterol content of theextracts. These results are similar to the selectivity observed whendry, ground DCF was used (see Tables 2 and 3). The sterol content of thefinal saponified oils were 15% and 17% when the extractant usedinitially was 20% and 25% water, respectively.

TABLE 4 A preliminary study of the relationship between water contentand the ethanol-extractable components in DCF on a 100 g scale. Wt % ofTLC Observations (most % Water saponified abundant to least abundantSample in ethanol sample lipids) 111A-4195 5 0.8 TAG, FFA, St, St-F,St-FA 111C-4195 15 0.8 FFA, TAG, St, St-F, St-FA 111D-4195 20 0.4 FFA,TAG, St, St-F, St-FA 111E-4195 25 0.4 FFA, St, ST-F, TAG, St-FA

TABLE 5 GC results for saponified, silylated DCF extracts obtained usingdifferent ratios of ethanol/water. % Water in Total FFA, Sample ethanolwt % Total Sterols, wt % 111A-4195 5 48 11 111C-4195 15 46 12 111D-419520 48 15 111E-4195 25 30 17

Example 3 Agitated Batch Extraction of DCF with Ethanol/Water

Since the DCF is not ground, extraction efficiency was lower thananticipated for complete corn fiber oil extraction. Agitation is one wayto increase extraction efficiency. For this series of pilot-scaleexperiments, a Microferm fermentor was used.

A. Using 80/20 Ethanol/Water

Sample #37-4233AR was prepared by extracting 750 g of dry DCF at 50° C.with 80/20 ethanol/water by stirring (500 rpm) in a Microferm fermentorfor two hours. The material was vacuum filtered while hot to remove thesolids. The extract was saponified using 10% wt/v KOH for two hours at50° C. The saponification reaction was quenched with concentrated HCl to˜pH1 and the volume was reduced by simple vacuum distillation. Hexaneextraction of the concentrated extract left many of the impuritiesbehind resulting in a 2.1% wt sample/wt of original DCF, but only a 24%recovery of material after saponification. Sample #37-4233AR contained60 wt % free fatty acids and 20 wt % total sterols. Despite an excess ofbase that generated salt upon quenching, the ash content was only 0.3%wt/wt. The water from the saponification step for Sample #37-4233AR wasfound to be low in amino acids, carbohydrates, and lipids.

A second 80/20 ethanol/water extraction resulted in Sample #93-4233AR.Dry, unground DCF (1000.0 g) was placed in the microferm fermentor andcovered with 10.0 L of 80/20 Ethanol/water at 50° C. for 2 hours withagitation (1000 rpm). The solid material was filtered using cheesecloth,and the filtrate (5.8 L) was cooled to room temperature. The filtratewas saponified using 1% wt/v NaOH at 50° C. for 2 hours with 500 rpmagitation before quenching to pH 4.0 with phosphoric acid. The ethanolwas removed with vacuum distillation to a volume of ˜1.8 L. Extractionwith hexanes (2 L), drying with magnesium sulfate, and distillation ofthe solvent resulted in Sample #93-4233AR (6.09 g; 0.6% wt sample/wtDCF). Total sterols for Sample #93-4233AR was 16.31 wt %. This number isonly slightly lower than that found in Sample #37-4233AR. The resultssuggest that 1% base during saponification, as was used to prepareSample #93-4233AR, is nearly as effective at saponification as was the10% base utilized in preparing Sample #37-4233AR.

B. Using 95/5 Ethanol/Water.

DCF (750.7 g dry) was extracted with 95% ethanol for 2 hours at 50° C.in the Microferm Fermentor with agitation. Vacuum filtration recovered6.4 L and resulted in a loss of 10.2% of the dry DCF mass. The volume ofthe extract was reduced to 2.15 L and aliquots were utilized in avariety of subsequent experiments.

Sample 50A-4233AR resulted from 300 mL of the extract being extractedwith hexanes. The hexane layer was back-extracted with both 1M NaOH and20% (v/v) H₂SO₄ to remove nonlipid impurities. The hexane layer wasdried using MgSO₄ and the solvent removed using simple vacuumdistillation. The mass of the resulting sample was 2.36 g and itcontained 5.95 wt % total sterol despite not undergoing saponificationprior to analysis. Approximately 75% of the total sample expected (14%by weight after saponification of the theoretical 3% oil from thestarting DCF=3.14 g) was recovered in a single, hot extraction withethanol/water. The 95/5 extract, with only acid and base washes, had aminimum of 6 wt % free sterol, since higher wt % sterols were possiblevia the degradation of fatty acid and ferulate esters.

Sample 60-4233AR resulted from 720 mL of the extract being refluxed for2 hours with 1% (wt/v) KOH. The reaction was quenched to pH˜1.5 andextracted with hexanes. The hexane layer was dried using MgSO₄ and thesolvent removed using simple vacuum distillation. The semi-crystallinesample mass was 0.52 g, a mere 7% of the theoretical oil expectation.This low overall yield was consistent with a large loss of nonlipidmaterial that becomes water soluble during treatment with base. Sample60-4233AR contains 55 wt % total sterol.

C. Exploring Milder Saponification Conditions

The experiments were begun with 95% ethanol/5% water extract of DCF(1130 mL of 50-4233AR) which should result in ˜0.8 g of saponified oil.Sodium hydroxide (1.3 g; 0.1 wt %) was added and the whole reactionmixture was transferred into the Parr Reactor. After 15 minutes of rapidbubbling of nitrogen gas, a 100 psi head pressure was initiated. Heatingto 245° C. began and was maintained for an hour. It was necessary toperiodically release pressure from the reactor in order to keep it below1000 psi. The reactor was cooled using 25° C. water and disassembled inthe hood. The contents were transferred to a 2 L Erlenmeyer flask andthe reactor was rinsed with new ethanol (2×50 mL). The pH was adjustedusing 85% phosphoric acid to ˜6.9. A slight precipitate formed prior toethanol removal by distillation. The reduced volume was extracted withhexanes. The hexane layer was dried with magnesium sulfate, filtered,and distilled to produce Sample 88-4233AR. Analysis of the productindicated 12.9% total sterols and 35% free fatty acids. TLC verifiedthat there are both St-FA and TAG present in the sample that are part ofthe complete mass balance. No St-F ester could be detected by TLC,despite what appears to be an incomplete reaction. It also appeared thatthe low base, high temperature and pressure reaction was not aseffective at degrading nonlipid material as 1% base at 50° C. for twohours.

Example 4 Using 1% Base During the Extraction Process

Dry DCF that had been partially de-oiled using 95/5 ethanol/water(50CF-4233AR; 100.2 g) was suspended in water (650 mL) and ethanol (100mL) in the presence of 1% (wt/v) KOH. The mixture was boiled on a hotplate for two hours. After limited cooling and the addition of coolwater (200 mL), ethanol (3 L to form 75/25 ethanol/water) was added toprecipitate the solubilized carbohydrates. Vacuum filtration resulted inrecovery of 3.4 L of solvent. The fiber was re-dried and found to havelost 16.2% (wt/wt) during the extraction. The filtrate was acidified topH 2 and extracted once with equal volumes of hexanes. The hexane layerwas dried using MgSO₄ and the solvent removed using simple vacuumdistillation to produce the semi-crystalline Sample 64-4233AR. Sample64-4233AR (1.54 g; 1.54 wt % based on deoiled DCF) contained 7.75 wt %total sterol and reinforces the fact that a single, batch extraction ofDCF results in incomplete phytosterol extraction. Sample 64H2O-4233AR,the water layer, contained 2.0 wt % ferulic acid and about 10% less ofcoumaric acid. The total elemental phosphorus in Sample 64H2O-4233AR was14 ppm.

Example 5 Extraction of Pretreated DCF Acid and ThermochemicallyPre-treated CF

Drying of a sample of corn fiber that was pretreated by heating at 130°C. for 30 minutes in the presence of 0.8 wt % acid (to remove all thestarch and hemicellulose) resulted in a mass of 81.54 g. The wholeportion was extracted with 95% ethanol (650 mL) for 2 hours at 50° C.with gentle agitation. The fiber was removed by hot vacuum filtration.The residue was re-dried and found to be 14% lighter than the initialdry mass. Base (6.3 g NaOH) was added to the filtrate and the resultingsolution was incubated at reflux for 2 hours. Quenching with sulfuricacid to pH˜2 was followed by hexane extraction. The hexane layer wasback-extracted with distilled water to ensure removal of the salts anddried with magnesium sulfate overnight. When the hexanes were removed,Sample 70-4233AR accounted for 7.08 g or 8.7% wt sample/wt of thestarting thermochemically treated corn fiber. The combined water layers,Sample 70H2O-4233AR, contained 19 ppm elemental phosphorus and a totalof 0.4 wt % ferulic acid. Coumaric acid was present at 47 ppm. Sample70-4233AR had 6.13 wt % total sterols. These results suggest thatremoval of starch and hemicellulose reduce the ferulic acid contentsignificantly, but appear to have a negligible impact on the phytosterolcontent of ethanol-extractable, saponified material.

Summary of Results from Examples 1-5

Table 6 details the conditions and results of the larger scaleextractions. The range of phytosterol content is 6-55 wt % in the finaloil samples. The only sample reported below that was not saponified wasSample 50A. Sample 50A came from the same initial corn fiber extract asboth Sample 60-4233AR and Sample 88-4233AR. With 6 wt % for 50A, 13 wt %for 88, and 55 wt % for 60, it appears that the saponificationconditions can make a significant difference in the final oil. Theoptimal saponification appears to occur with 1% base. When theextraction solvent is 20% water in ethanol (samples 37 and 90) thevariation in sterol content of the final, saponified oil is not asdramatic. This is due to the fact that the farther the water content inthe extractant is from 0%, the less sterol fatty acid esters are removedfrom the corn fiber.

Corn fiber that has been pretreated by initial oil removal (i.e., Sample64) or by thermochemical (i.e., Sample 70) do not show significantdifferences in the sterol content of the final, saponified oil. Thesterol content of Samples 64 (7.75 wt %) and 70 (6.13 wt %) is similarto the unsaponified extract 50A (5.95 wt %).

TABLE 6 Compilation of extraction and saponification conditions withresults from Examples 1-5. Sterol FFA % Water in Content Content EthanolSaponification Yield (Wt % (Wt % (Wt % Sample extraction Conditionssample/fiber) sample/oil) sample/oil) 37-4233AR 20 10% base; 2 hrs 0.520 60 93-4233AR 20 1% base; 2 hrs 0.6 16.31 57.67 50-4233AR 5 None;washed extract 2.25 5.95 0.83 with acid & base 60-4233AR 5 1% base; 2hrs 0.21 55 9.2 88-4233AR 5 0.1% base; 250 C.; — 12.9 35 1000 psi; 1 hr64-4233AR 25 with 1% During extraction; 2 1.54 7.75 27.4 base; deoiledhrs DCF 70-4233AR 5; acid & 10% base; 2 hrs 8.7 6.13 73.5 thermo. DCF

Having now fully described the present invention in some detail forpurposes of clarity of understanding, it will be obvious to one ofordinary skill in the art that the same can performed by modifying orchanging the invention within a wide and equivalent range of conditions,formulations, and other parameters without affecting the scope of theinvention or any specific embodiment thereof, and that suchmodifications or changes are intended to be encompassed within the scopeof the appended claims.

All publications, patents, and patent applications metnioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patent,or patent application was specifically and individually indicated to beincorporated by reference.

1. A method of extracting phytosterols from corn fiber comprising: a)combining corn fiber having one or more phytosterols with a greensolvent selected from the group consisting of water, ethanol, isopropylalcohol, ethyl lactate, acetone, butanol, isoamyl alcohol, ethylacetate, and any combinations thereof to produce a mixture comprisingsaid green solvent and said corn fiber, wherein said mixture furthercomprises one or more dissolved phytosterols from said corn fiber; b)removing said corn fiber from said mixture of a) to produce an extract;c) saponifying said extract of b) to produce a saponified extract; d)removing said green solvent from said saponified extract of c) toproduce a saponified material; e) combining said saponified material ofd) with an organic solvent thereby extracting water soluble componentsfrom said saponified material of d); f) removing said organic solvent ofe) from said saponified material of e); and g) recovering saidsaponified material of f) to obtain a composition comprisingphytosterols.
 2. The method of claim 1, wherein said compositionconsists essentially of free phytosterols and oil.
 3. The method ofclaim 2, wherein said oil consists essentially of free fatty acids. 4.The method of claim 1, wherein said composition comprises phytosterolsselected from the group consisting of beta-sitosterol, sitostanol,campesterol, campestanol, stigmasterol, stigmastanol, brassicasterol,sterol glucosides, sterol esters, sterol fatty acid esters, sterolferulates, sterol ferulate esters, and combinations thereof.
 5. Themethod of claim 1, wherein said corn fiber is prepared by wet-millingcorn.
 6. The method of claim 1, wherein said corn fiber is destarched.7. The method of claim 5, wherein an amount of water is removed fromsaid wet-milled corn fiber prior to the combining of said corn fiberwith said green solvent.
 8. The method of claim 7, wherein said cornfiber comprises up to about 65% water.
 9. The method of claim 8, whereinsaid corn fiber comprises about 20% water.
 10. The method of claim 1,wherein said green solvent consists of a mixture of water and ethanol.11. The method of claim 10, wherein said green solvent consists of about80% ethanol and about 20% water.
 12. The method of claim 1, whereinagitation is performed on said mixture.
 13. The method of claim 12,wherein said agitation is performed at a speed of up to 1000 rpm. 14.The method of claim 13, wherein said agitation is performed at a speedfrom 250 to 1000 rpm.
 15. The method of claim 14, wherein said agitationis performed at a speed of about 500 rpm.
 16. The method of claim 1,wherein heating is performed on said mixture.
 17. The method of claim16, wherein said heating occurs at a temperature from 25° C. to 50° C.18. The method of claim 17, wherein said heating occurs at a temperatureof 50° C.
 19. The method of claim 1, wherein said saponifying isperformed by adding an amount of base that is from 0.1% weight/volume to10% weight/volume of said extract of b).
 20. The method of claim 19,wherein said saponifying is performed by adding an amount of base thatis about 1% weight/volume of said extract of b).
 21. The method of claim20, wherein said saponifying is performed at a temperature of about 250°C. and at a pressure of up to 1000 psi.
 22. The method of claim 1,wherein said green solvent is removed from said saponified material ofd) by simple distillation.
 23. The method of claim 1, wherein said greensolvent is removed from said saponified material of d) by rotaryevaporation.
 24. The method of claim 1, wherein said green solvent isremoved from said saponified material of d) by simple vacuumdistillation.
 25. The method of claim 1, wherein said organic solvent isremoved by simple distillation.
 26. The method of claim 1, wherein saidorganic solvent is removed by rotary evaporation.
 27. The method ofclaim 1, wherein said organic solvent is removed by simple vacuumdistillation.
 28. A phytosterol composition extracted by the method ofclaim
 1. 29. The method of claim 1, wherein said organic solvent ishexane.